The electrically stimulated guinea pig longitudinal muscle -myenteric plexus (LMMP) - preparation has been developed as a bioassay procedure for enkephalinase activity of mouse and guinea pig brain. Met enkephalin was the substrate used. Highest enzyme activity was found in the 20,000xg pellet. D-phenylalanine (DPA), bacitracin and puromycin were found to inhibit enkephalinases; inhibitory potency parallels analgesic activity in mice. Using radioimmune assay (RIA), we have shown that met enkephalin is depleted by analgesic doses of these compounds as well as by morphine. Injected radioactive DPA is taken up slowly into mouse brain and remains in the brain for extended periods of time. Uptake into brain of young mice is much greater than old mice. DPA appears to be incorporated directly into brain protein. Using puromycin in the bath, we have shown by RIA that tetanic stimulation of the LMMP does not cause an increase in the release of met enkephalin over resting amounts. Indomethacin administered to mice along with chronic morphine prevented the development of tolerance to the analgesic action of the opiate.